ABSTRACT Therapeutic strategies are needed to improve the efficacy of immune checkpoint blockade (ICB) therapy in pancreatic ductal adenocarcinomas (PDAC). PDACs have an increased reliance on the DNA damage response (DDR) for mitigating oncogene-induced replication stress and, the DDR regulates innate immunity via regulation of cGAS/STING/TBK1-mediated detection of cancer DNA. ATM is the apical kinase in the DDR and the target of small molecule inhibitors in clinical development. Furthermore, ionizing radiation stimulates the cGAS/STING/TBK1 innate immune pathway to modulate immune responses in a type 1 interferon (T1IFN)- dependent fashion that are required for the synergy of radiation with ICB. Therefore, the central hypothesis of this proposal is that a novel direct link between ATM and innate immune sensing pathways can be leveraged therapeutically in combination with radiation to enhance the tumoral T1IFN pathway and improve ICB efficacy in otherwise poorly immunogenic PDACs. This hypothesis will be tested in three specific aims: Aim 1 will define the immunologic consequences of ATM inhibition in combination with radiation and the mechanisms by which ATM affects innate immunity in PDAC. In this aim we will assess the contributions of cytoplasmic DNA (1A), ATM substrates (1B), and pattern recognition receptor pathway signaling (1C) to immune endpoints such as T1IFN-mediated signaling, PD-L1 expression, and T cell-mediated killing (1D). Aim 2 will investigate the immune contribution to the sensitivity of ATM depleted PDAC tumors to the combination of radiation and PD-L1 therapy. Our preliminary data suggest that ATM has both tumor and host immune-dependent mechanisms (i.e. T1IFN secretion) that influence the sensitivity of tumors to combined anti-PD-L1 and radiation. We will determine the contribution of tumoral and host T1IFN signaling to the sensitivity of ATM-deficient tumors to combined anti-PD-L1 and radiation therapy (2A) as well as the immune consequences (2B). We hypothesize that the therapeutic advantages of ATM deficiency will be diminished in T1IFNR deficient tumor cells and hosts since tumoral T1IFN production likely increases tumor immunosurveillance through both tumor and host-dependent mechanisms. In Aim 3 we will develop a therapeutic strategy combining ATM inhibitors and radiation with anti-PD-L1 in PDAC. Our preliminary data show that pharmacologic ATM inhibition activates the immune pathway. We will determine the efficacy of the clinical candidate ATM inhibitor AZD0156 in combination with anti-PD-L1 and the optimal radiation dose/fractionation schema in syngeneic PDAC tumors and autochthonous PDAC tumors in genetically engineered mouse models (3A). We will also develop pharmacodynamic biomarkers that will be predictive of the therapeutic efficacy of ATM inhibition and radiation in combination with anti-PD-L1 (3B). Completion of these aims will define a new connection between ATM, radiation and innate immunity that will be leveraged therapeutically to extend the efficacy of ICB to PDAC which is highly relevant to the mission of the NIH.